Understanding how function and substrate specificity are diversified within the members of an enzyme superfamily represents a key question in protein chemistry with profound implications for evolution, protein design and for the engineering of useful enzymes for biomedical applications. Here we present a comprehensive and highly integrated experimental research program for exploring how to interconvert substrate specificity among enzyme superfamily members exhibiting a low degree of amino acid sequence identity. As part of this work, entirely new chimeric enzymes, derived from the combinatorial assembly of subdomains from parental sequences, will be isolated from highly diverse libraries. Enzyme isolation and the interrogation of the libraries for function and substrate specificity will be accomplished by virtue of quantitative, ultra-high throughput screening that capitalizes predominantly on single cell, flow cytometric assays. Chimeric enzymes exhibiting desired profiles of catalytic activity and substrate selectivity will be crystallized, high resolution structures will be obtained where possible and finally, the catalytic mechanism of the enzymes will be analyzed in detail. As part of this study we will examine how the combinatorial assembly of protein subdomains can be employed to interconvert the specificity of serine proteases (elastase and chymotrypsin) and to transform the specificity of the human glutathione S transferase to that of the rat enzyme. In parallel we will explore the limits imposed by the decreasing amino acid sequence identity for the two parental genes selected for combination and seek to overcome these limits by mutagenesis. The gene pairs will be chosen from the extensive family of dihydrofolate reductase sequences allowing a systematic variation from 42% to 28% sequence identity--in all cases below that of classical DNA shuffling. Consequently, the proposed studies will help delineate the secondary structural elements and specific amino acids that dictate: (a) the cleavage specificity in trypsin proteases; (b) recognition of electrophile substrates in glutathione conjugation by GST enzymes and finally (c) protein folding and catalytic activity in dihydrofolate reductase. The generation of enzymes having novel substrate specificity profiles distinct from either parent will also be investigated. Finally, but perhaps equally importantly, this work will validate a unique, highly interdisciplinary approach for the exploration and deeper understanding of enzyme function. [unreadable] [unreadable]